AAV gene therapy for treating nervous system diseases

Abstract

A recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant or functional fragment thereof, wherein the recombinant AAV expression vector comprises a neuron-specific promoter, is provided. A pharmaceutical composition comprising the rAAV expression vector and its use are further provided.

Description

Background Art

[0001] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease and motor neuron disease (MND), is a specific disease that causes the death of neurons that control voluntary muscles. Some also use the term "motor neuron disease" for the group of conditions where ALS is most common. ALS is characterized by muscle stiffness, muscle fasciculations, and a gradually worsening weakness due to a decrease in muscle size. As a result, speaking, swallowing, and ultimately breathing become difficult. In 90% - 95% of cases, the cause is unknown. Approximately 5 - 10% of cases are inherited from a person's parents. Approximately half of these genetic cases are due to four specific genes, SOD1, TDP - 43, FUS, and C9orf72. Currently, there are only three clinical drugs for the treatment of ALS. One is riluzole, which was approved over 20 years ago. The results of clinical trials have shown that riluzole can only extend the survival of patients by a few months. The second, edaravone, seems to be effective only in the early stages of the disease and delays the progression of the disease by more than two months, but in fact, it can accelerate the progression of the disease in the middle and late stages. Patients in the AMX0035 treatment group showed only a slight statistically significant and clinically significant improvement, extending the survival period of patients with the disease by 4.9 months. For drugs in clinical trials, there have been few breakthroughs so far, and they have only delayed the progression of the disease by less than five months. Drug development for ALS has been difficult for decades. Currently, gene therapy is becoming one of the most promising treatments for neurodegenerative diseases including ALS. Gene therapy has led to more molecules being connected to the development of new drugs. However, almost all current ALS gene therapy strategies are limited to targeting ALS patients with familial mutations, while there is no viable solution for sporadic ALS patients. Oxidative stress is a common pathological process in all ALS patients, and therefore, the development of gene therapy drugs targeting oxidative stress is a potentially viable way to treat both familial ALS and sporadic ALS.

[0002] TRIM72 is a tripartite motif (TRIM) family protein consisting of a ring finger, a B-box motif, a coiled-coil region, and a C-terminal PRYSPRY domain. It is involved in the fascia repair process and is related to the insulin signaling pathway. It is also involved in cardiac protection against ischemia / reperfusion (IR) injury. Full-length TRIM72 has been reported to act as a potential target for ALS by ubiquitinating mutant FUS protein. However, it is necessary to further investigate the effect of TRIM72 on protecting neurons and to further develop different types of TRIM72 proteins / gene products.

Summary of the Invention

[0003] The present disclosure provides a recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant or functional fragment thereof. The rAAV expression vector has one or more of the following characteristics: (1) it can highly express the TRIM72 protein in vivo, for example, in neurons; (2) it can effectively protect neurons; (3) it can reduce oxidative stress; (4) it can treat, prevent, and / or alleviate nervous system diseases.

[0004] In one aspect, the present application provides a recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant or functional fragment thereof, wherein the recombinant AAV expression vector comprises a neuron-specific promoter.

[0005] In some embodiments, the neuron-specific promoter comprises a human-derived promoter.

[0006] In some embodiments, the promoter is selected from the group consisting of an excitatory neuron-specific promoter, a cerebral cortex and hippocampal excitatory neuron-specific promoter, a short neuron-specific promoter, a dopaminergic neuron-specific promoter, a glutaminergic neuron-specific promoter, a GABAergic neuron-specific promoter, a cholinergic neuron-specific promoter, and a serotonergic neuron-specific promoter.

[0007] In some embodiments, the promoter is selected from the group consisting of human synapsin (hSyn), calcium / calmodulin-dependent kinase IIa (CamKIIa), c-fos, methyl CpG-binding protein 2 (Mecp2), neuron-specific enolase (NSE), somatostatin (SST), human vesicular GABA (gamma-aminobutyric acid) transporter (hVGAT), choline acetyltransferase (ChAT), serotonin transporter (SERT), and tyrosine hydroxylase (TH).

[0008] In some embodiments, the serotype of the AAV vector is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh, AAVDJ, and AAVhull.

[0009] In some embodiments, the AAV is single-stranded AAV (ssAAV) or self-complementary AAV (scAAV).

[0010] In some embodiments, the TRIM72 protein is a human TRIM72 protein.

[0011] In some embodiments, the TRIM72 protein comprises the full-length TRIM72 protein.

[0012] In some embodiments, the TRIM72 protein comprises the wild-type TRIM72 protein.

[0013] In some embodiments, the human TRIM72 protein comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0014] In some embodiments, the TRIM72 protein comprises a TRIM72 cleavage protein.

[0015] In some embodiments, the TRIM72 cleavage protein comprises the PRYSPRY domain of the TRIM72 protein or a functional fragment thereof.

[0016] In some embodiments, the PRYSPRY domain comprises the amino acid site of 278aa-470aa of the TRIM72 protein.

[0017] In some embodiments, the PRYSPRY domain comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0018] In some embodiments, the TRIM72 cleavage protein further comprises the coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

[0019] In some embodiments, the TRIM72 cleavage protein does not comprise the coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

[0020] In some embodiments, the coiled-coil domain comprises the amino acid site of 135aa-232aa of the TRIM72 protein.

[0021] In some embodiments, the coiled-coil domain comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0022] In some embodiments, the TRIM72 cleavage protein further comprises the B box domain of the TRIM72 protein or a functional fragment thereof.

[0023] In some embodiments, the TRIM72 cleavage protein does not contain the B-box domain of the TRIM72 protein or a functional fragment thereof.

[0024] In some embodiments, the B-box domain contains the amino acid site of 86aa-117aa of the TRIM72 protein.

[0025] In some embodiments, the B-box domain contains the amino acid sequence shown in SEQ ID NO: 4.

[0026] In some embodiments, the TRIM72 cleavage protein further contains the ring finger domain of the TRIM72 protein or a functional fragment thereof.

[0027] In some embodiments, the TRIM72 cleavage protein does not contain the ring finger domain of the TRIM72 protein or a functional fragment thereof.

[0028] In some embodiments, the ring finger domain contains the amino acid site of 14aa-56aa of the TRIM72 protein.

[0029] In some embodiments, the ring finger domain contains the amino acid sequence shown in SEQ ID NO: 3.

[0030] In some embodiments, the TRIM72 cleavage protein contains the amino acid sequence shown in any one of SEQ ID NOs: 6, 7, 8, 9, and 11.

[0031] In some embodiments, the TRIM72 protein or a variant or functional fragment thereof contains an amino acid mutation at position C14.

[0032] In some embodiments, the TRIM72 protein or a variant or functional fragment thereof contains the amino acid mutation C14A.

[0033] In some embodiments, the TRIM72 protein or a variant or functional fragment thereof does not contain an amino acid mutation at position C242.

[0034] In some embodiments, the rAAV expression vector is used to protect neurons by reducing oxidative stress.

[0035] In some embodiments, the rAAV expression vector is used to prevent or treat nervous system diseases.

[0036] In some embodiments, the rAAV expression vector is used to prevent or treat ALS or stroke.

[0037] In another aspect, the present application provides a host cell comprising the rAAV expression vector.

[0038] In another aspect, the present application provides a pharmaceutical composition comprising the rAAV expression vector or the host cell and a pharmaceutically acceptable adjuvant.

[0039] In another aspect, the present application provides a method for protecting neurons of a subject, comprising administering an effective amount of the rAAV expression vector, the host cell, and / or the pharmaceutical composition to a subject in need thereof.

[0040] In another aspect, the present application provides a method for preventing and / or treating a nervous system disease, comprising administering an effective amount of the rAAV expression vector, the host cell, and / or the pharmaceutical composition to a subject in need thereof.

[0041] In some embodiments, the nervous system disease includes ALS or stroke.

[0042] In another aspect, the present application provides the use of the rAAV expression vector, the host cell, and / or the pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a nervous system disease.

[0043] In some embodiments, the neurological disorder includes ALS or stroke.

[0044] Further embodiments and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present disclosure. As will be understood, the present disclosure is capable of other and different embodiments, and some of the details thereof are capable of modification in various obvious respects without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0045] Incorporation by reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0046] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained from the following detailed description, which illustrates exemplary embodiments in which the principles of the invention are utilized, and from the appended drawings (also referred to herein as "figures" and "FIGs"). BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0048] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, numerous modifications, changes, and substitutions may occur to those skilled in the art. It should be understood that various alternative forms to the embodiments described herein can be used.

[0049] In this application, the term "adeno-associated virus vector" generally refers to nucleic acids derived from any AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 serotypes, or any other virus or serotype homologous to the capsid protein sequence of an AAV serotype. The term "recombinant adeno-associated virus" or "rAAV" refers to an infectious replication-deficient virus composed of an AAV protein shell encapsulating a nucleic acid molecule of interest, with AAV ITRs adjacent to one or both sides. As used herein, reference to a specific AAV serotype means an AAV having at least one capsid protein of that AAV serotype. For example, the term "AAV9" refers to an AAV having at least one AAV serotype 9 capsid protein.

[0050] In this application, the term "TRIM72 protein" can be used interchangeably with the term "MG53" protein and generally includes a TRIM72 protein or its variants, functional fragments, analogs, homologs. The TRIM72 protein may include a ring finger, B-box motif, coiled-coil region, and / or C-terminal PRYSPRY domain. For example, the ring finger domain may include the amino acid site of 14aa - 56aa of the TRIM72 protein or its functional fragment. For example, the B-box domain may include the amino acid site of 86aa - 117aa of the TRIM72 protein or its functional fragment. For example, the coiled-coil domain may include the amino acid site of 135aa - 232aa of the TRIM72 protein or its functional fragment. For example, the PRYSPRY domain may include the amino acid site of 278aa - 470aa of the TRIM72 protein. This term may also include TRIM72 proteins derived from any known species having the TRIM72 protein.

[0051] In the present application, the term "truncated protein" generally refers to a protein having one or more amino acid deletions as compared to the full-length protein. For example, a truncated protein may include a major functional fragment of the protein. For example, truncated proteins include, but are not limited to, variants, functional fragments, analogs, and homologs thereof.

[0052] In the present application, "amino acid mutation Xn" refers to an amino acid mutation occurring at the nth amino acid residue X of an amino acid sequence, where n is a positive integer and X is an abbreviation for any amino acid residue, in the amino acid sequence shown in SEQ ID NO: 2. For example, "amino acid mutation C14" refers to an amino acid substitution occurring at the amino acid residue C corresponding to the 14th position in the amino acid sequence shown in SEQ ID NO: 2.

[0053] The amino acid mutations in the present application may be non-conservative mutations. The non-conservative mutations may involve changing the amino acid residues in the target protein or polypeptide in a non-conserved manner, for example, replacing an amino acid residue having a specific side chain size or a specific characteristic (e.g., hydrophilicity) with an amino acid residue having a different side chain size or a different characteristic (e.g., hydrophobicity).

[0054] The amino acid substitution may be a conservative substitution. The conservative substitution may involve changing the amino acid residues in the target protein or polypeptide in a conserved manner, for example, replacing an amino acid residue having a specific side chain size or a specific characteristic (e.g., hydrophilicity) with an amino acid residue having the same or a similar side chain size or the same or a similar characteristic (e.g., still hydrophilic). Such conservative substitutions generally do not have a significant effect on the structure or function of the resulting protein. In the present application, an amino acid sequence variant that is a variant of a fusion protein, a fragment thereof, or a variant thereof that undergoes one or more amino acid substitutions may include conservative amino acid substitutions that do not significantly change the structure or function of the protein.

[0055] As an example, the following mutual substitutions between amino acids in each group can be considered conservative substitutions in the present application. Group of amino acids with non-polar sides: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine.

[0056] Group of uncharged amino acids with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0057] Group of negatively charged amino acids with polar side chains: aspartic acid and glutamic acid.

[0058] Group of positively charged basic amino acids: lysine, arginine, and histidine.

[0059] Group of amino acids having a phenyl: phenylalanine, tryptophan, and tyrosine.

[0060] In the present application, the term "vector" generally refers to a vector containing a recombinant polynucleotide that includes an expression control sequence efficiently linked to the nucleotide sequence to be expressed. The vector contains cis-acting elements sufficient for expression. Other elements for expression may be provided by the host cell or in an in vitro expression system. The vector can include all expression vectors known in the art that can be incorporated into recombinant polynucleotides, including cosmids, plasmids (e.g., naked or encapsulated in liposomes), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0061] In the present application, the term "encoding" generally refers to the inherent property of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA or mRNA that acts as a template for the synthesis of other multimers and macromolecules in a biological process, wherein the multimers and macromolecules have either a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence, and biological properties result therefrom. Thus, when transcription and translation of an mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. A nucleotide sequence can be referred to as a gene or cDNA encoding a protein or other product, both for the coding strand that is identical to the mRNA sequence and usually provided in the sequence listing, and for the non-coding strand that is used as a template for transcription of the gene or cDNA. In the present application, the term "coding element" generally refers to a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence encoding a protein.

[0062] In the present application, the terms "host cell", "cell", and "host" are used interchangeably and generally refer to a plasmid or vector that can or has contained a nucleic acid molecule of the present application, or can express an individual cell, cell line or cell culture of a protein, fragment or variant thereof of the present application. The host cell can include progeny of a single host cell. Due to natural, accidental or deliberate mutations, progeny cells and the original parental cells are not necessarily identical in form or genome as long as they can express the protein or fragment thereof of the present application. The host cell can be obtained by transfecting cells in vitro with a vector of the present application.

[0063] In the present application, the term "treating" generally refers to delaying or improving the progression, severity, and / or duration of a proliferative condition, or improving one or more symptoms (e.g., one or more distinguishable symptoms) of a proliferative condition as a result of administration of one or more therapies.

[0064] In the present application, the term "subject" generally refers to any human or non-human animal. The term "non-human animal" can include all vertebrates, such as mammals and non-mammals, such as non-human primates, goats, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

[0065] In addition to the specific proteins and nucleotides referred to herein, the present application may also include functional variants, derivatives, analogs, homologs, and fragments thereof.

[0066] The term "functional variant" refers to a polypeptide that has an amino acid sequence that is substantially the same as a naturally occurring sequence or is encoded by a substantially the same nucleotide sequence and can have one or more activities of the naturally occurring sequence. In the context of the present application, a variant of any given sequence refers to a sequence in which the specific sequence of residues (either amino acid residues or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially maintains at least one endogenous function. Variant sequences can be obtained by addition, deletion, substitution, modification, exchange, and / or mutation of at least one amino acid residue and / or nucleotide residue present in a naturally occurring protein and / or polynucleotide, as long as the original functional activity is maintained. In the present application, the term "derivative" generally refers to a polypeptide or polynucleotide of the present application that includes any substitution, mutation, modification, exchange, deletion, and / or addition from / to one (or more) amino acid residues of the resulting polypeptide or polynucleotide, as long as the resulting polypeptide or polynucleotide substantially maintains at least one of its endogenous functions.

[0067] In this application, the term "analogue" generally refers to, with respect to a polypeptide or polynucleotide, any mimetic of the polypeptide or polynucleotide, i.e., a compound having at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics. Generally, amino acids can be substituted as long as the modified sequence substantially maintains the required activity or ability, for example, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 or more) amino acids can be substituted. Amino acid substitutions can include the use of non-naturally occurring analogues. The proteins or polypeptides used in this application may also have deletions, insertions or substitutions of amino acid residues, and the amino acid residues may undergo silent changes, resulting in a functionally equivalent protein. Intentional amino acid substitutions can be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphoteric properties of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid. Positively charged amino acids include lysine and arginine. Amino acids containing uncharged polar head groups with similar hydrophilic values include asparagine, glutamine, serine, threonine and tyrosine.

[0068] In this application, the term "homolog" generally refers to an amino acid sequence or a nucleotide sequence that has a certain homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" may be equivalent to "identity" of the sequences. Homologous sequences may include amino acid sequences that are at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the target sequence. Generally, homologs contain the same active site as the target amino acid sequence, etc. Homology may be considered based on similarity (i.e., amino acid residues having similar chemical properties / functions), or homology may be expressed in terms of sequence identity. In this application, a sequence having percentage identity at any of the sequence numbers of the recited amino acid sequences or nucleotide sequences refers to a sequence having percentage identity over the full length of the recited sequence number. To determine sequence identity, sequence alignment can be performed by various methods known to those skilled in the art, for example, by using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters suitable for alignment, including any algorithms necessary to achieve an optimal alignment in the full-length sequences being compared.

[0069] In this application, the term "about" generally refers to varying within a range of 0.5% - 10% above or below the specified value, for example, varying within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

[0070] In this application, the term "comprising" usually means including, containing, having, or encompassing. In some cases, it also refers to the meaning of "being present" or "consisting of".

[0071] In the present application, the term "not containing" generally refers to excluding a specific behavior, structure, or the possibility of a structure. For example, "A does not contain B" generally means excluding the possibility of B occurring in A.

[0072] Recombinant adeno-associated virus (rAAV) expression vector In one aspect, the present application provides a recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant thereof or a functional fragment thereof, wherein the recombinant AAV expression vector comprises a neuron-specific promoter.

[0073] In this application, rAAV may contain an AAV genome or its derivative, and / or an AAV capsid protein or its derivative. In this application, rAAV can be chimeric AAV, shuffled AAV, or capsid-modified AAV. In this application, the AAV genome or AAV capsid protein can be derived from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh, AAVDJ, and AAVhull. In this application, rAAV can be a hybrid AAV (e.g., AAV-DJ, AAV-DJ / 8, or AAV-DJ / 9). In this application, rAAV can be developed by directed evolution and / or rational design (e.g., AAV 7m8 or AAV-PHP.B). In this application, rAAV can contain one or more capsid mutations (e.g., one or more capsid mutations among Y444F, Y500F, Y730F, Y252F, Y272F, Y700F, Y704F, and T491V, or AAV2 having corresponding mutations to different AAV serotypes (e.g., AAV2 / 8 (Y733F), AAV2 (Y444F + Y500F + Y730F), and AAV2 (quadY-F + T-V))). In this application, the serotype of rAAV can be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Anc80, rh10, and ShH10. In this application, the rAAV vector can be selected from the group consisting of AAV2 / 5, AAV2 / 8, AAV2 / 8 (Y733F), AAV2 (Y444F + Y500F + Y730F), AAV2 / 1, AAV2 / 4, AAV2 / 9, AAV2 / 6, AAV2 / 7, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, Anc80, AAV7m8, AAV-DJ, ShH10, AAV-PHP.B, or their hybrids, derivatives, or variants. In this application, the rAAV vector can be a single-stranded AAV vector or a self-complementary AAV (scAAV) vector.

[0074] In some embodiments, the serotype of the AAV vector can be AAV9.

[0075] In the present application, the AAV packaging system can be used for the delivery of a gene encoding the TRIM72 protein or a variant or a functional fragment thereof. For example, the AAV packaging system can be a triple plasmid system. For example, the AAV packaging system can include pAAV-ITR containing a gene encoding the TRIM72 protein or a variant or functional fragment thereof, a serotype vector pRepCapX, and a helper vector pADHelper.

[0076] In the present application, the neuron-specific promoter can include a human-derived promoter.

[0077] In the present application, the promoter can include a common neuron-specific promoter. In the present application, the promoter can include an excitatory neuron-specific promoter. In the present application, the promoter can include a cerebral cortex and hippocampal excitatory neuron-specific promoter. In the present application, the promoter can include an inhibitory neuron-specific promoter. In the present application, the promoter can include a short neuron-specific promoter. In the present application, the promoter can include a dopaminergic neuron-specific promoter. In the present application, the promoter can include a glutaminergic neuron-specific promoter. In the present application, the promoter can include a GABAergic neuron-specific promoter. In the present application, the promoter can include a cholinergic neuron-specific promoter. In the present application, the promoter can include a serotonergic neuron-specific promoter.

[0078] In this application, the neuron-specific promoter can be selected from the group consisting of human synapsin (hSyn), calcium / calmodulin-dependent kinase IIa (CamKIIa), c-fos, methyl CpG-binding protein 2 (Mecp2), neuron-specific enolase (NSE), somatostatin (SST), human vesicular GABA (γ-aminobutyric acid) transporter (hVGAT), choline acetyltransferase (ChAT), serotonin transporter (SERT), and tyrosine hydroxylase (TH). For example, the promoter can be the Syn1 promoter. For example, the Syn1 promoter can contain the nucleotide sequence shown in SEQ ID NO: 12 or a variant thereof.

[0079] In this application, the rAAV expression vector contains a gene encoding a TRIM72 protein or a variant or functional fragment thereof.

[0080] In this application, the TRIM72 protein can include the full-length TRIM72 protein. In this application, the TRIM72 protein can include the wild-type TRIM72 protein. In this application, the human TRIM72 protein includes the amino acid sequence shown in SEQ ID NO: 2.

[0081] In this application, the TRIM72 protein can include a TRIM72 cleavage-type protein.

[0082] In this application, the TRIM72 cleavage-type protein includes a PRYSPRY domain or a functional fragment thereof. For example, the TRIM72 cleavage-type protein can include the amino acid site of 278aa-470aa of the TRIM72 protein. For example, the TRIM72 cleavage-type protein can include the amino acid sequence set in SEQ ID NO: 6.

[0083] In this application, the TRIM72 cleavage-type protein can further include other domains of the TRIM72 protein.

[0084] For example, the TRIM72 cleavage protein may include a PRYSPRY domain and a coiled-coil domain. For example, the TRIM72 cleavage protein may include a deletion of the B box domain and the ring finger domain of the TRIM72 protein. For example, the TRIM72 protein may include the amino acid sequence shown in SEQ ID NO: 11.

[0085] For example, the TRIM72 cleavage protein may include a PRYSPRY domain and a B box domain. For example, the TRIM72 cleavage protein may include a deletion of the coiled-coil domain and the ring finger domain of the TRIM72 protein.

[0086] For example, the TRIM72 cleavage protein may include a PRYSPRY domain and a ring finger domain. For example, the TRIM72 cleavage protein may include a deletion of the B box domain and the coiled-coil domain of the TRIM72 protein.

[0087] For example, the TRIM72 cleavage protein may include a PRYSPRY domain, a coiled-coil domain, and a ring finger domain. For example, the TRIM72 cleavage protein may include a deletion of the B box domain of the TRIM72 protein.

[0088] For example, the TRIM72 cleavage protein may include a PRYSPRY domain, a coiled-coil domain, and a B box domain. For example, the TRIM72 cleavage protein may include a deletion of the ring finger domain of the TRIM72 protein.

[0089] For example, the TRIM72 cleavage protein may include a PRYSPRY domain, a ring finger domain, and a B box domain. For example, the TRIM72 cleavage protein may include a deletion of the coiled-coil domain of the TRIM72 protein.

[0090] In this application, the TRIM72 protein or a fragment thereof may include its variants. For example, the TRIM72 protein may include one or more amino acid mutations as compared to the corresponding wild-type sequence.

[0091] In this application, the TRIM72 protein may include an amino acid mutation at position C14. For example, the amino acid mutation may be C14A.

[0092] According to the research of the applicant, the amino acid cysteine at position 242 is important for the oligomer formation of the TRIM72 protein. Substitution of the amino acid C242 may block the protective function of neurons. Therefore, the amino acid substitution at position C242 (for example, C242A) may not be included in the TRIM72 protein in this application.

[0093] In this application, the rAAV expression vector may be used to protect neurons by reducing oxidative stress.

[0094] In this application, the rAAV expression vector may be used to prevent or treat nervous system diseases. For example, this application may be used to prevent and / or treat ALS.

[0095] Host cell, pharmaceutical composition In another aspect, this application provides a cell (for example, a host cell) that can include the rAAV expression vector of this application.

[0096] In some embodiments, the host cell is a bacterial cell, an E. coli cell, a plant cell, an insect cell, or a mammalian cell. In some embodiments, the cell is a somatic cell or a stem cell. In some embodiments, the rAAV expression vector is delivered to the host cell such that Cas encoded by the nucleic acid molecule is expressed intracellularly.

[0097] In another aspect, this application provides a pharmaceutical composition including the rAAV expression vector or the host cell and a pharmaceutically acceptable adjuvant.

[0098] In some embodiments, pharmaceutically acceptable adjuvants can include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants, etc.

[0099] In the present application, the pharmaceutical composition can be formulated with pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients according to the conventional technical means in the art, for example, according to the procedures of Remington: The Science and Practice of Pharmacy, 19th edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995.

[0100] In the present application, the composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or the drug is administered via a subcutaneous depot.

[0101] In the present application, the pharmaceutical composition can be used to protect neurons. For example, the composition of the present application can inhibit or delay the onset or progression of a nervous system disease (such as ALS or stroke), and / or can reduce and / or stabilize the disease state.

[0102] The pharmaceutical composition of the present application can include a therapeutically effective amount of the rAAV expression vector. The therapeutically effective amount is the dose required to prevent and / or treat (at least partially treat) the disease (such as ALS or stroke) and / or any complications thereof in a subject having the disease and / or at risk of the disease.

[0103] Preparation, methods and uses In another aspect, the present application provides a method for protecting a target neuron, the method comprising administering an effective amount of an rAAV expression vector, host cell, and / or pharmaceutical composition to a subject in need thereof.

[0104] In another aspect, the present application provides a method for preventing and / or treating a neurological disease, the method comprising administering an effective amount of an rAAV expression vector, host cell, and / or pharmaceutical composition to a subject in need thereof.

[0105] In another aspect, the present application provides the use of an rAAV expression vector, host cell, and / or pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a neurological disease.

[0106] In another aspect, the present application provides an rAAV expression vector, host cell, and / or pharmaceutical composition for use in preventing and / or treating a neurological disease.

[0107] In the present application, the neurological disease may include a neurodegenerative disease or other diseases.

[0108] In the present application, the neurological disease includes ALS or stroke. For example, the stroke may be an ischemic stroke.

[0109] Example The following examples are set forth to provide a complete disclosure and description of how to make and use the invention to those skilled in the art and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise noted, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations, e.g., bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; nt, nucleotide; i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); rpm, revolutions per minute; may be used.

[0110] Materials and Methods Animals ALS-causing mutant SOD1 generated by using cDNA encoding human TDP43 with an N-terminal Flag tag having the A315T mutation G93AIn this project, the ALS mouse model SOD1-G93A with overexpression of hTDP43-A315T was used, while age-matched wild-type (WT) mice were used as controls for each experiment. The animal facility at Tsinghua University has received full accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) since 2014. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Tsinghua University based on the Guide for the Care and Use of Laboratory Animals (8th Edition, NHR). All mice were housed in a barrier facility with individually ventilated cages (up to 6 mice per cage) at Tsinghua University. The mice were maintained on a 12 / 12 hour light / dark cycle, at 22 - 26 °C, and 40 - 70% humidity, with free access to sterile pellet food and water. The cages were checked daily to ensure the health of the animals. The body weight was regularly evaluated to ensure no weight loss. When animals were used in the study, the inventors followed the 3R (Replacement, Refinement, or Reduction) rules.

[0111] Behavioral test For the open field behavioral test, a single animal was placed in the center of an open field area (60×60 cm), and tracked using the TopScan behavioral analysis system (CleverSys, USA) with multiple parameters including the total distance, average speed, and distance moved in the central area at 10-minute intervals. Rotarod performance was measured by an automated system (Med Associates Inc.). Briefly, the animal was placed on an accelerating spindle (5 - 40 rpm) for 5 minutes / trial and 3 trials / day in consecutive trials. A 20-minute rest was set between each trial. The fall time from the spindle was automatically calculated by the system when the mouse fell off the spindle within 5 minutes. The stay time was calculated by subtracting the fall time from 5 minutes, and the average value of the stay time from 3 consecutive trials per day was used for statistical analysis. Mice were assigned to different experimental groups according to age, genotype, and treatment. Behavioral assays were performed randomly in different groups. The adhesive removal test was used to evaluate neurological dysfunction after stroke, which is a sensitive method for evaluating sensorimotor impairment in focal cerebral ischemia mice. Briefly, training sessions were conducted until the mouse could remove the adhesive dot on its own foot within 12 seconds before the surgical procedure. The animals were tested by researchers blinded to the experimental group 3, 5, or 7 days after ischemia. Both the time taken to sense the stimulus and the time taken to remove the tape were measured.

[0112] Evaluation of experimental endpoints As described above, the body weight of the mice was measured three times a week and evaluated for debilitation. The mouse reached the experimental endpoint when it could not stand up within 30 seconds after turning its back.

[0113] Immunoblot The total protein content in cells or tissues was homogenized in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.25% sodium deoxycholate, 0.1% SDS, 1% NP-40, supplemented with a complete protease inhibitor mixture; Bimake B15001), and then the lysate was incubated on ice for 30 minutes. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was extracted, mixed with SDS loading buffer, and then incubated at 95°C for 10 minutes. Next, the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, it was blocked with 5% non-fat milk at room temperature for 1 hour. Subsequently, it was incubated overnight at 4°C with diluted primary antibodies including GAPDH (Ameribio), TUBULIN (Ameribio), TRIM72 antibody (a kind gift from Dr. Jianjie Ma), TSG101 (Abcam), ITGAV (Abcam), and H3 (Abcam). Then, the membrane was incubated with an HRP-conjugated secondary antibody at room temperature for 1 hour. Images were analyzed using Fiji ImageJ to obtain the integrated intensity.

[0114] Exosome purification The protocol for purifying exosomes from 100 ml of the supernatant of 293FT cells included two steps: ultrafiltration and polyethylene glycol (PEG) precipitation. First, the collected supernatant was poured into a centrifuge tube and centrifuged at 3000 x g for 20 minutes to remove cell debris. Then, the supernatant was filtered once through a 0.45 μm filter. Then, a new Amicon ULTRA-15 ultrafiltration tube washed with PBS or autoclaved water was taken. Next, the supernatant was immediately added to the ultrafiltration tube and centrifuged at 3000 x g for 5 - 10 minutes. The filtrate was discarded, and the supernatant was added continuously, and centrifuged at 3000 x g for 5 - 10 minutes until all the supernatant was introduced into the ultrafiltration tube. Then, the concentrate was transferred to a 50 mL centrifuge tube. An isolation reagent was added to the concentrate, and the sample was completely mixed by vortexing or pipetting. Finally, the sample was left at 2 - 8 °C overnight. The next day, the sample was centrifuged at 10,000 x g for 1 hour at 4 °C. When the supernatant was discarded, the exosomes were in the pellet.

[0115] Middle cerebral artery occlusion / reperfusion (MCAO / R) model Male C57BL / 6J mice weighing 25 - 28 g were used in this study. The mice were anesthetized with 1.2% tribromoethanol. The necks of the mice were shaved and disinfected with povidone iodine solution, then a midline incision was made to expose the carotid artery, and then the common carotid artery (CCA) and external carotid artery (ECA) were ligated using 6-0 silk suture to permanently occlude the blood flow. A silicon-coated nylon suture (MSMC23B100PK50, RWD) was introduced into the internal carotid artery (ICA) through the arteriotomy site of the CCA stump until it reached the base of the skull, as indicated by feeling moderate resistance. The remaining loose monofilament suture was gently tightened around the CCA to prevent backflow leakage, then the wound was covered with a cotton ball, and the mice were transferred to a warming pad to maintain body temperature. After 1 hour, the silicon-coated nylon suture was gently withdrawn, and the monofilament suture was tightly ligated to avoid postoperative bleeding. Finally, the wound was closed with absorbable suture.

[0116] Oxygen-glucose deprivation (OGD) In OGD, the medium was exchanged with HEPES-buffered glucose-free medium containing (in mmol / L) 154 NaCl, 5.6 KCl, 2.3 CaCl2, 1.0 MgCl2, 3.6 NaHCO3, 5 HEPES, pH 7.4. OGD was induced by incubating N2a cells in this medium (multiplicity of infection (MOI): 10,000 vg / cell) for 24 hours after scAAV9 infection, and then placing the cells in an anaerobic chamber with an atmosphere of 95% N2 and 5% CO2 at 37°C. The glucose-free medium was returned to Neurobasal medium, and the cultures were incubated in a normal cell culture incubator for 22 hours or for the reoxygenation period (reperfusion, R) specifically indicated in individual experiments for 2 hours, 4 hours, or 6 hours to terminate OGD. Control cells were treated similarly except that they were not exposed to OGD.

[0117] Plasmid and lentiviral vector A DNA fragment corresponding to full-length TRIM72 was amplified from a mouse cDNA library by PCR and inserted into the pCMV-N-3×Flag expression vector between the SalI site and the XhoI site using a seamless cloning kit (Beyotime) to generate Flag-tagged TRIM72. A series of Flag-tagged TRIM72 domain deletions: Δring domain (deletion of domains 14 - 69); ΔB box domain (deletion of 81 - 122 aa); Δcoiled-coil domain (deletion of 135 - 232 aa); ΔPRYSPRY domain (deletion of 278 - 470 aa) constructs were generated from the full-length TRIM72 expression vector.

[0118] For the TRIM72 stable expression cell line constructs, a series of Flag-tagged TRIM72 mutants or domain deletions or single-domain DNA fragments were amplified from the corresponding TRIM72 expression vector by PCR and inserted into the pLJM1-EGFP lentiviral vector between the BsrG I site and the EcoR I site to generate a series of Flag-tagged TRIM72 mutants or domain deletions or single domains fused with EGFP in the lentiviral vector.

[0119] A lentivirus expressing a Flag-tagged TRIM72 construct was generated from the corresponding EGFP fused with a Flag-tagged TRIM72 vector.

[0120] Cell culture, lentivirus packaging and lentivirus infection HEK293FT cells were maintained in DMEM (Invitrogen) containing 10% fetal bovine serum (Gemini) in a cell incubator (37 °C, 5% CO2). For lentivirus packaging, HEK293FT cells were seeded into the growth medium of three 10-cm culture dishes. When reaching approximately 90% confluence, the cells were co-transfected with VSVG (10 μg), pxPAX2 (15 μg) and the pLJM1-EGFP lentivirus vector or pLentiCRISPRv2 (Addgene) or pLenticas9-Blast (Addgene) (20 μg) using PEI (Sigma) according to the manufacturer's instructions. Five to six hours after transfection, the medium was replaced with medium containing fresh growth medium. The medium was collected 72 hours after transfection and centrifuged at 20,000 rpm at 4 °C for 2 hours. After centrifugation, the lentivirus was concentrated into a pellet. The lentivirus was resuspended in 100 μl of DPBS and stored at -80 °C.

[0121] HEK293FT cells or Hela cells were infected with the indicated lentivirus. Three days after infection, the infected cells were selected with 2 μg / ml puromycin or 10 μg / ml blasticidin according to the plasmid containing resistance for at least one week. Puromycin or blasticidin selected cells were applied for further analysis.

[0122] Cell viability assay Cell viability was evaluated using CCK-8. The cells were seeded at 1.5×10 per well for arsenite treatment 3 cells, or 7×10 per well for H2O2 treatment 3Cells were seeded in 96-well plates at a density of cells. Arsenite (Sigma) was added to each well at a concentration of 0.125 mM, 0.25 mM, or 0.5 mM, and after incubation at 37 °C for 2 hours, the cells were washed. In the case of the H2O2 treatment experiment, scAAV9 infection (estimated multiplicity of infection (MOI): 10,000 vg / cell) was performed for 24 hours 17 hours after cell seeding. Then, H2O2 was added to each well at a concentration of 300 μM, and after incubation at 37 °C for 1 hour, the cells were washed. After treating the cells with arsenite or H2O2, a total of 10 μL of CCK-8 solution (Yeasen) was added to each well. After further incubation at 37 °C for 2 hours, the optical density (OD) value of each well was measured using a microplate reader with an excitation wavelength of 450 nm. The cell viability of 293FT was calculated. The experiment was repeated at least 3 times to obtain the average value.

[0123] Example 1 AAV Packaging and AAV Vector Constructs The AAV packaging system is a commonly used triple plasmid system. By simultaneously transfecting three plasmids into mammalian cells (e.g., HEK293), all the components necessary for AAV packaging can be expressed in this cell and assembled into virus particles. The inventors used a modified triple plasmid system from PackGene (Guangzhou PackGene Biotech Co., Ltd.). This system consists of three plasmids: pAAV-ITR containing the target gene, the serotype vector pRepCapX, and the helper vector pADHelper. The target vector pAAV-ITR contains a eukaryotic promoter and other components required for high-level gene expression in mammalian gene cells when the foreign sequence is cloned into the polylinker site (MCS). The vector also contains the AAV inverted terminal repeat sequence (ITR) that guides virus replication and packaging. PRepCapX contains the AAV rep and CAP genes encoding the replication protein and the virus capsid protein. Stabilization of the rep and CAP gene expression levels is an important step to obtain the desired high-titer virus product. pADHelper contains a collection of adenovirus genes VA, E2A, and E4 that are essential for the cell production of high-titer virus.

[0124] To construct the pAAV-ITR plasmids for TRIM72 expression, the inventors used both self-complementary AAV (scAAV) and single-stranded (ss) AAV vectors. Compared to the ssAAV9 vector, the scAAV9 vector is 10- to 100-fold more efficient in transfection and expression, but can only package up to 2.2 kb of foreign DNA. Furthermore, scAAV persists as a stable episome in non-dividing cells, and studies have reported stable transgene expression over many years. Figure 1 shows different pAAV-ITR vectors containing TRIM72 (SEQ ID NO: 2) or a control gene. The human synapsin 1 (hSyn1) promoter is a neuron-specific promoter. The GfaABC1D promoter, a truncated GFAP promoter, is an astrocyte-specific promoter. Each construct contains, for ssAAV, the 5’ ITR and 3’ ITR, or for scAAV, the 5’ ITR and 3’ ITR-Δtr, a neuron (hSyn1) or astrocyte (GfaABC1D) specific promoter, mouse TRIM72 (mTRIM72) or human TRIM72 (hTRIM72) cDNA, the SV40 polyadenylation signal (SV40 polyA) or the bovine growth hormone polyadenylation signal (BGH polyA), followed by the presence or absence of the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE).

[0125] To deliver sufficient amounts of the AAV vector to the nervous system and provide sufficient levels of gene expression, the following (non-limiting) routes of administration can be used: intracerebral administration, intrathecal administration, intravenous administration, aerosol administration, and intranasal, intramuscular, subcutaneous, intradermal, rectal, and other parenteral routes of administration. If desired, the routes of administration can be combined.

[0126] Example 2 Therapeutic effect of TRIM72 in an ssAAV vector on an ALS animal model To examine the therapeutic effect of TRIM72 in AAV vectors against ALS, classical ALS animal models, SOD1-G93A transgenic mice (The Jackson Lab: stock number 002726) and hTDP43-A315T transgenic mice (The Jackson Lab: stock number 010700) were used here. Mutant SOD1 is an important genetic factor leading to the etiology of ALS. SOD1-G93A transgenic mice expressing a high copy number of mutant SOD1 develop adult-onset degeneration of spinal motor neurons and progressive motor impairment, resulting in paralysis and death. TDP43-A315T transgenic mice express a mutant human TAR DNA-binding protein cDNA with an amino acid substitution associated with familial ALS. Hemizygous mice develop a progressive and lethal neurodegenerative disease associated with both ALS with ubiquitin aggregates and frontotemporal lobar degeneration.

[0127] In the first experiment, ssAAV(PHP.eB)-EGFP-TRIM72 was used as the experimental group and AAV(PHP.eB)-EGFP was used as the control. As shown in Figure 2, according to the AAV(PHP.eB) administration procedure, an AAV vector at a dose of 10 11 vg / mouse was injected into SOD1-G93A mice, or 3×10 11The AAV vectors of vg / mouse doses were injected into TDP43-A315T mice by retro-orbital intravenous injection respectively. The SOD1-G93A model mice showed a significant tendency of weight loss at 130 days of age and continued until the end of the experiment. However, the weight loss was relatively small in the EGFP-TRIM72 group compared to the AAV(PHP.eB)-EGFP group during disease progression. This indicated that AAV-TRIM72 had a moderate but significant protective effect against weight loss due to disease progression in male mice (Figure 3). Furthermore, to determine the effect of AAV-TRIM72 on the motor ability of SOD1-G93A, open field tests and rotarod tests of locomotor movement were conducted during the treatment of AAV therapy. As the disease progressed, the total spontaneous movement distance gradually decreased due to the progressive death of motor neurons. AAV-TRIM72 was suggested to have a partial protective effect against the decline in the spontaneous motor ability of SOD1-G93A mice (Figure 4). In addition, the treatment of AAV-TRIM72 significantly preserved motor skills as evaluated by the rotarod performance test, and there was a significant difference in the latency to fall in mice over 120 days of age (Figure 5). Finally, the survival data indicated that AAV-TRIM72 had only a mild effect on the survival of mice (Figure 6). Furthermore, the survival period of TDP43-A315T mice was significantly extended after administration of TRIM72 by ssAAV. The median survival period of the ssAAV(PHP.eB)-hSyn1-EGFP-TRIM72 treatment group was about 133 days, while the median survival period of the ssAAV(PHP.eB)-EGFP treatment group was about 117 days. It was suggested that AAV-TRIM72 had a protective effect on the lifespan extension of TDP43-A315T mice (Figure 9A).

[0128] To examine the expression of EGFP-TRIM72 in target regions including the motor cortex and spinal cord, mouse tissues were sectioned and stained for the GFP-tag, neurons, and motor neurons (Figure 7). In the motor cortex and spinal cord of SOD1-G93A transgenic mice, infected neurons and motor neurons were observed. The infection efficiency of ssAAV(PHP.eB)-hSyn1-EGFP-TRIM72 was calculated according to the ratio of infected neurons to all neurons. The efficiency was approximately 20.95% in the cortex and approximately 29.8% in the spinal cord. Further analysis also showed a positive correlation between the infection efficiency and the animal survival days in the AAV(PHP.eb)-TRIM72-GFP treatment group but not in the AAV(PHP.eb)-GFP treatment group in both SOD1-G93A mice and TDP43-A315T mice (Figures 8 and 9B).

[0129] To characterize the expression of EGFP-TRIM72 in other non-neural tissues, the ssAAV(PHP.eB)-hSyn1-EGFP-TRIM72 construct was injected into wild-type C57BL / 6 mice at a dose of 10 11 vg / mouse, and tissues were collected for immunoblotting 10 days after expression. No obvious expression was detected in non-neural tissues such as the heart, kidney, liver, lung, muscle, and spleen (Figure 11). When the hSyn1 promoter was used, the expression of EGFP-Trim or GFP was mainly distributed in the cortex and spinal cord, and when the GfaABC1D promoter was used, it was mainly distributed in astrocytes (Figure 10).

[0130] Example 3 Therapeutic effect of TRIM72 in scAAV vectors on ALS animal models To achieve better therapeutic performance, a higher dose (2×10 14scAAV9-hSyn1-TRIM72 (vg / kg body weight) was used in the following experiment (the sequence of the Syn1 promoter is as shown in SEQ ID NO: 12). A vehicle was used as a control. An enhanced therapeutic effect was observed in the scAAV9-hSyn1-TRIM72 treatment group. The improved motor function was demonstrated by rotarod behavior (Figure 12). Furthermore, scAAV9-hSyn1-TRIM72 treatment sufficiently delays disease progression in SOD1-G93A mice as indicated by a 15% weight loss (Figure 13A). Survival data also showed that a dose of 2×10 14 vg / kg body weight of scAAV9-hSyn1-TRIM72 could significantly extend the median survival time of SOD1-G93A mice (Figure 13B). GfaABC1D (SEQ ID NO: 13) is a glial cell-specific promoter. When another efficacy analysis was performed in hTDP43-A315T mice, a significant therapeutic effect was also observed in the group treated with scAAV(PHP.eB)-hSyn1-TRIM72 at a dose of 8×10 12 vg / kg body weight, but not in the group treated with ssAAV(PHP.eB)-GfaABC1D-TRIM72 at a dose of 10 13 vg / kg body weight (Figure 14). The median survival time of the scAAV(PHP.eB)-hSyn1-TRIM72 treatment group was approximately 124 days, while that of the control group was approximately 115 days. However, the ssAAV(PHP.eB)-GfaABC1D-TRIM72 treatment group was less effective than the control group, with a median survival time of only 103 days. Riluzole was used as a reference, which was the same as the control group in terms of median survival time.

[0131] These results are also consistent with the inventors' previous findings. In particular, compared to the control group, FUS-R521C knock-in mice (C / C) did not exhibit a severe ALS phenotype and had no significant differences in behavior as shown by the rotarod test or survival curve (Figure 15). Western analysis showed that TRIM72 was overexpressed in the cortex and spinal cord of mice (C / C) and rats (C / C) (Figures 16-17). Further analysis demonstrated that TRIM72 was specifically upregulated in neurons of FUS-R521C knock-in mice (C / C), but remained at low levels in wild-type (+ / +) (Figure 18). Furthermore, loss of function of Trim72 (- / -) was able to significantly shorten the stay time in the rotarod test and the lifespan of FUS-R521C knock-in mice (Figure 15). Therefore, upregulation of TRIM72 in neurons is an important factor for FUS-R521C knock-in mice not to exhibit a severe ALS phenotype. Therefore, specific expression of TRIM72 in neurons was able to achieve effective treatment for ALS and other neuronal damage diseases.

[0132] Example 4 Protective effect of truncated TRIM72 in scAAV vector in cultured cells The TRIM72 protein contains a ring finger motif, B box domain, coiled coil domain, and PRYSPRY domain (Figure 19). To investigate which domain of TRIM72 plays a role in protection from oxidative stress, full-length TRIM72 or domain-disrupted TRIM72 mutants were constructed and stably overexpressed in 293FT cell lines by lentiviral infection and puromycin selection (Figure 20). CCK-8 was used to measure cell viability after arsenite treatment. The inventors found that coiled coil domain- and PRYSPRY domain-disrupted TRIM72 mutants abolished the protective effect of TRIM72, while ring domain- or B box domain-disrupted TRIM72 mutants retained a protective effect equivalent to that of the wild type (Figure 21).

[0133] To further investigate which important domains of the TRIM72 protein are required to protect neurons, different domain-disrupted TRIM72s were constructed and overexpressed simultaneously in N2a cell lines by scAAV9 infection (Figure 22). CCK-8 was used to measure cell viability after H2O2 treatment. Indeed, cell viability decreased after H2O2 treatment (Figure 23). Notably, overexpression of the coiled PRYSPRY or PRYSPRY single domain was sufficient to protect cells from oxidative stress, while overexpression of the coiled-coil single domain was not, suggesting that either coiled PRYSPRY cleavage or the PRYSPRY single domain may induce a protective effect (Figure 23).

[0134] Example 5 scAAV-TRIM72 prevents and alleviates brain injury and behavioral dysfunction in MACO / R mice Here, the middle cerebral artery occlusion / reperfusion (MCAO / R) model was used as a disease model of ischemic stroke. According to the administration procedure shown in Figure 24, scAAV9-hSyn1-TRIM72 vector at a dose of 4×10 14 vg / kg body weight was injected into mice by retro-orbital intravenous injection. Quantification of TTC staining showed that the size of the infarct area in mice of the scAAV-TRIM72 treatment group decreased compared to that of the control group (data not shown). Furthermore, scAAV-TRIM72 pretreatment was demonstrated to prevent and alleviate behavioral disorders in the motor and sensory activity tasks of neurological tests and adhesive removal tests 5 or 7 days after MCAO / R, where the MCAO / R.scAAV-TRIM72 pretreatment group had better behavioral scores (Figure 25A) and was able to detect the presence of adhesive tape in a shorter time after MCAO / R (Figure 25B).

[0135] Example 6 scAAV-TRIM72 prevents cell death after OGD N2a cells experience an increase in oxidative stress and cell death when subjected to OGD pretreatment. Investigate and confirm whether TRIM72 plays an important role in protection after OGD treatment. Full-length TRIM72 was overexpressed in the N2a cell line by scAAV9 infection (estimated multiplicity of infection (MOI): 10,000 vg / cell) (Figure 26), and cell viability was measured using CCK-8. Indeed, cell viability decreased after OGD treatment, but overexpression of TRIM72 increased cell viability (Figure 26).

[0136] Example 7. TRIM72 protects cells from oxidative stress, while other TRIM proteins do not To investigate whether other TRIM proteins can also induce a protective effect from oxidative stress, TRIM40 was overexpressed in the N2a cell line by scAAV9 infection (estimated multiplicity of infection (MOI): 10,000 vg / cell) (Figure 22). Cell viability after H2O2 treatment was measured using CCK-8. Notably, treatment with TRIM72 but not TRIM40 induced a protective effect from oxidative stress, demonstrating that only TRIM72 can protect cells from oxidative stress, while other TRIMs cannot (Figure 23).

[0137] Example 8. Important sites of the TRIM72 protein that protect cells from oxidative stress A series of Flag-tagged TRIM72 mutants: C14A (cysteine at position 14 substituted by alanine); C242A (cysteine at position 242 substituted by alanine) were generated from the wild-type TRIM72 construct by site-directed mutagenesis.

[0138] Previous studies have determined that TRIM72 senses changes in the oxidative environment and forms an oligomeric complex to complete membrane repair. The cysteine residue (C242) plays an important role in TRIM72 oligomer formation. Furthermore, the cysteine residue (C14) is important for TRIM72 E3 ligase activity. Mutation of C242 to alanine (C242A) blocked the TRIM72 protective effect, whereas its E3 ligase-inactive mutant (C14A) retained a protective effect equivalent to that of the wild type (Figure 27). It can be concluded that TRIM72 protects cells from oxidative stress and mainly depends on its oligomerization rather than its E3 ligase activity.

[0139] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not to be construed in a limiting sense. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the present invention. Further, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend on various conditions and variables. It should be understood that various alternative forms may be used in practicing the present disclosure. Accordingly, the present invention is intended to cover any such alternative, modified, variant, or equivalent forms. The following claims define the scope of the present invention, and it is intended that the methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

**Claim 1** A recombinant adeno-associated virus (rAAV) expression vector comprising a gene encoding a TRIM72 protein or a variant or functional fragment thereof, wherein the recombinant AAV expression vector comprises a neuron-specific promoter, the rAAV expression vector. **Claim 2** The rAAV expression vector according to claim 1, wherein the neuron-specific promoter comprises a human-derived promoter. **Claim 3** The rAAV expression vector according to any one of claims 1 to 2, wherein the promoter is selected from the group consisting of an excitatory neuron-specific promoter, a cerebral cortex and hippocampal excitatory neuron-specific promoter, a short neuron-specific promoter, a dopaminergic neuron-specific promoter, a glutaminergic neuron-specific promoter, a GABAergic neuron-specific promoter, a cholinergic neuron-specific promoter, and a serotonergic neuron-specific promoter. **Claim 4** The rAAV expression vector according to any one of claims 1 to 3, wherein the promoter is selected from the group consisting of human synapsin (hSyn), calcium / calmodulin-dependent kinase IIa (CamKIIa), c-fos, methyl CpG-binding protein 2 (Mecp2), neuron-specific enolase (NSE), somatostatin (SST), human vesicular GABA (gamma-aminobutyric acid) transporter (hVGAT), choline acetyltransferase (ChAT), serotonin transporter (SERT), and tyrosine hydroxylase (TH). **Claim 5** The rAAV expression vector according to any one of claims 1 to 4, wherein the serotype of the AAV vector is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh, AAVDJ, and AAVhull. **Claim 6** The rAAV expression vector according to any one of claims 1 to 5, wherein the rAAV is single-stranded AAV (ssAAV) or self-complementary AAV (scAAV). **Claim 7** The rAAV expression vector according to any one of claims 1 to 6, wherein the TRIM72 protein is a human TRIM72 protein. **Claim 8** The rAAV expression vector according to any one of claims 1 to 7, wherein the TRIM72 protein comprises the full-length TRIM72 protein. **Claim 9** The rAAV expression vector according to any one of claims 1 to 8, wherein the TRIM72 protein comprises a wild-type TRIM72 protein.

10. The rAAV expression vector according to any one of claims 7 to 9, wherein the human TRIM72 protein comprises the amino acid sequence shown in SEQ ID NO:

2.

11. The rAAV expression vector according to any one of claims 1 to 10, wherein the TRIM72 protein comprises a TRIM72 cleavage-type protein.

12. The rAAV expression vector according to claim 11, wherein the TRIM72 cleavage-type protein comprises the PRYSPRY domain of the TRIM72 protein or a functional fragment thereof.

13. The rAAV expression vector according to claim 11, wherein the PRYSPRY domain comprises the amino acid site of 278aa-470aa of the TRIM72 protein.

14. The rAAV expression vector according to any one of claims 12 to 13, wherein the PRYSPRY domain comprises the amino acid sequence shown in SEQ ID NO:

6.

15. The rAAV expression vector according to any one of claims 12 to 14, wherein the TRIM72 cleavage-type protein further comprises the coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

16. The rAAV expression vector according to any one of claims 12 to 14, wherein the TRIM72 cleavage-type protein does not comprise the coiled-coil domain of the TRIM72 protein or a functional fragment thereof.

17. The rAAV expression vector according to any one of claims 15 to 16, wherein the coiled-coil domain comprises the amino acid site of 135aa-232aa of the TRIM72 protein.

18. The rAAV expression vector according to any one of claims 15 to 17, wherein the coiled-coil domain comprises the amino acid sequence shown in SEQ ID NO:

5.

19. The rAAV expression vector according to any one of claims 12 to 18, wherein the TRIM72 cleavage-type protein further comprises the B-box domain of the TRIM72 protein or a functional fragment thereof.

20. The rAAV expression vector according to any one of claims 12 to 18, wherein the TRIM72 cleavage-type protein does not comprise the B-box domain of the TRIM72 protein or a functional fragment thereof.

21. The rAAV expression vector according to any one of claims 19 to 20, wherein the B box domain contains the amino acid site of 86aa-117aa of the TRIM72 protein.

22. The rAAV expression vector according to any one of claims 19 to 21, wherein the B box domain contains the amino acid sequence shown in SEQ ID NO:

4.

23. The rAAV expression vector according to any one of claims 12 to 22, wherein the TRIM72 truncated protein further contains the ring finger domain of the TRIM72 protein or a functional fragment thereof.

24. The rAAV expression vector according to any one of claims 12 to 22, wherein the TRIM72 truncated protein does not contain the ring finger domain of the TRIM72 protein or a functional fragment thereof.

25. The rAAV expression vector according to any one of claims 23 to 24, wherein the ring finger domain contains the amino acid site of 14aa-56aa of the TRIM72 protein.

26. The rAAV expression vector according to any one of claims 23 to 25, wherein the ring finger domain contains the amino acid sequence shown in SEQ ID NO:

3.

27. The rAAV expression vector according to any one of claims 11 to 26, wherein the TRIM72 truncated protein contains the amino acid sequence shown in any one of SEQ ID NOs: 6, 7, 8, 9, and 11.

28. The rAAV expression vector according to any one of claims 1 to 27, wherein the TRIM72 protein or a variant or a functional fragment thereof contains an amino acid mutation at position C14.

29. The rAAV expression vector according to any one of claims 1 to 28, wherein the TRIM72 protein or a variant or a functional fragment thereof contains the amino acid mutation C14A.

30. The rAAV expression vector according to any one of claims 1 to 29, wherein the TRIM72 protein or a variant or a functional fragment thereof does not contain an amino acid mutation at position C242.

31. The rAAV expression vector according to any one of claims 1 to 30, which is used for protecting neurons by reducing oxidative stress.

32. The rAAV expression vector according to any one of claims 1 to 31, which is used for preventing or treating nervous system diseases.

33. The rAAV expression vector according to any one of claims 1 to 32, which is used for preventing or treating ALS or stroke.

34. A host cell comprising the rAAV expression vector according to any one of claims 1 to 33.

35. A pharmaceutical composition comprising the rAAV expression vector according to any one of claims 1 to 33 or the host cell according to claim 34 and a pharmaceutically acceptable adjuvant.

36. A method for protecting neurons of a subject, the method comprising the step of administering an effective amount of the rAAV expression vector according to any one of claims 1 to 33, the host cell according to claim 34, and / or the pharmaceutical composition according to claim 35 to a subject in need thereof.

37. A method for preventing and / or treating a neurological disease, the method comprising the step of administering an effective amount of the rAAV expression vector according to any one of claims 1 to 33, the host cell according to claim 34, and / or the pharmaceutical composition according to claim 35 to a subject in need thereof.

38. The method according to claim 37, wherein the neurological disease includes ALS or stroke.

39. Use of the rAAV expression vector according to any one of claims 1 to 33, the host cell according to claim 34, and / or the pharmaceutical composition according to claim 35 in the manufacture of a medicament for preventing and / or treating a neurological disease.

40. The use according to claim 39, wherein the neurological disease includes ALS or stroke.

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